Prions are unconventional, highly infectious agents, which are composed entirely of a protein that adopts an abnormal conformation. In mammals, prion-mediated infections are responsible for several devastating and invariably fatal neurodegenerative diseases, collectively known as transmissible spongiform encephalopathies. A hallmark of prion diseases is the presence of amyloids, which are also associated with the pathology of non-prion diseases, ranging from Alzheimer's and Huntington's disease to systemic amyloidosis. The broad and long-term research objective is to uncover the functional role of molecular chaperones in prion replication. Yeast provides an excellent paradigm to investigate the mechanism of prion replication. [PSI+] is a yeast prion that increases translational read-through of nonsense codons. Like mammalian prions, [PSI+] consist entirely of protein and is formed by self-replicating amyloid conformers of the evolutionary conserved translation termination factor Sup35p (eRF3). The inheritance and maintenance of [PSI+] are governed by Hsp104, a 600-kDa, ring-forming ATP-dependent, protein-remodeling machine, which cooperates with the Hsp70 chaperone system in prion replication and protein disaggregation. The objective of this research is to provide a detailed mechanistic understanding of the prion-remodeling and protein disaggregating activities of Hsp104 and its bacterial homolog ClpB. Three specific aims are proposed: 1) to determine the 3D structure of an Hsp104-substrate complex, 2) to investigate the synergistic interaction between Hsp104 and Hsp70/Hsp40, and 3) to elucidate the mechanism of protein disaggregation and prion replication by the Hsp104 bi-chaperone system. To address our research questions, we will use a multi-facet approach consisting of hybrid structural biology methods, proteomic and chemical biology techniques, and yeast genetics. The combination of these methods provides a powerful approach to yield new mechanistic insight into the structure-function relationship of this remarkable family of ATP-dependent molecular machines in order that this information might be exploited to engineer new nano-machines with novel biological activities with potential applications in biotechnology and nano-medicine.